共轭亚油酸对仔猪脂肪细胞凋亡的影响

发布时间：2018-03-20 09:33

本文选题：共轭亚油酸　切入点：脂肪沉积　出处：《西南大学》2015年硕士论文　论文类型：学位论文

【摘要】：共轭亚油酸(Conjugated linoleic acid, CLA)具有抑制动物脂肪沉积的作用,但其作用机制并不完全清楚。本论文结合动物营养学研究手段与分子生物学技术,从个体和分子水平系统探讨了CLA通过诱导脂肪细胞凋亡的方式来抑制猪的脂肪沉积,揭示其可能存在的作用机制。试验选用36头长白×荣昌仔猪,体重13.91+1.53kg,随机分为3个处理组,每个处理组3个重复,每个重复4头仔猪,分别饲喂含0%(对照组)、1%和2%CLA的日粮,试验持续30天。试验结束时,仔猪空腹12h后称重,随后从每个重复中选取2头体重适中的仔猪进行静脉采血,分离得血清,用以血液生化指标的测定。然后,仔猪放血屠宰,测量背部皮下脂肪厚度,剥离背部脂肪组织和腹部脂肪组织进行称重。分别取大块背最长肌和半腱肌,用以肌内脂肪含量测定。此外,每头仔猪分别取三份脂肪组织,分别用于形态学观察,Caspase-3、-8、-9等凋亡因子酶活分析,以及凋亡相关因子的基因和蛋白表达水平检测。主要试验结果如下：1.CLA显著降低了仔猪日均采食量(P0.05),而对料重比影响不显著(P0.05)。1%和2% CLA组分别使仔猪日均增重下降13.13%(P0.05)和7.95%(P0.05)。2.日粮添加CLA显著减少了仔猪的背部脂肪沉积量(P0.05)。并且随着CLA添加水平的提高,背膘厚降低。利用生化分析仪检测血清,结果发现CLA对仔猪血清中的葡萄糖、甘油三酯、低密度脂蛋白胆固醇和高密度脂蛋白胆固醇等生化指标影响不明显(P0.05),但显著降低了总胆固醇含量(P0.05)。3.通过对脂肪组织切片的免疫荧光染色观察发现CLA处理组仔猪的背部脂肪细胞表现出明显的凋亡迹象,包括染色质聚集、核浓缩、DNA断裂等凋亡特征,尤其是2% CLA组脂肪细胞凋亡特征更为明显。酶活检测结果表明日粮添加CLA使仔猪背部脂肪组织中的凋亡因子Caspase-3、-8和-9的活力均显著升高(P0.05)。荧光定量PCR和western blotting检测结果表明CLA处理下调了抑凋亡因子Bcl2的mRNA和蛋白表达,升高了促凋亡因子Bax和P53的mRNA及蛋白表达,同时增加Cyt-C的mRNA表达并且促进Cyt-C蛋白从线粒体释放到细胞质中。Caspase-3的mRNA表达上调和剪切型的Caspase-3蛋白水平的增加说明凋亡执行因子Caspase-3被激活并介导了脂肪细胞凋亡的发生。另一方面,CLA处理后还提高了脂肪细胞中TNF-α及其受体TNFR1的mRNA表达以及蛋白水平,而它们是死亡受体凋亡通路的标志因子。以上这些结果提示线粒体通路和死亡受体通路都参与了CLA诱导的猪背部脂肪细胞凋亡。以上试验结果表明,日粮补充CLA能够通过诱导脂肪细胞凋亡的方式来减少仔猪的背部脂肪沉沉,且线粒体通路和死亡受体通路都参与了凋亡信号的传递。
[Abstract]:Conjugated linoleic acid (CLA) can inhibit fat deposition in animals, but its mechanism is not completely clear. The mechanism of CLA inhibiting fat deposition in pigs by inducing adipocyte apoptosis was studied systematically at individual and molecular level. 36 Landrace 脳 Rongchang piglets were used in the experiment. The body weight was 13.91 1.53kg, and was randomly divided into three treatment groups, three replicates in each treatment group and four piglets per replica. The piglets were fed diets containing 0% and 2 CLA respectively. The experiment lasted 30 days. At the end of the experiment, the piglets were weighed after 12 hours fasting. Then two moderately weight piglets were selected from each repeat for venous blood collection, and the serum was isolated for the determination of blood biochemical indexes. Then, the piglets were bloodletting and slaughtered, and the thickness of subcutaneous fat on the back was measured. In addition, three portions of fat tissue were taken from each piglet to measure the fat content in the muscle, and the longissimus dorsi muscle and the semitendinosus muscle were taken to measure the intramuscular fat content. It was used to observe the activity of Caspase-3, caspase-3, caspase-8, and other apoptotic factors, respectively. The main results were as follows: 1. CLA significantly reduced the daily feed intake of piglets (P0.05), but had no significant effect on the feed / weight ratio (P0.05). 1% and 2% CLA groups decreased the average daily weight gain of piglets by 13.13 and P0.05, respectively. Dietary supplementation of CLA significantly reduced back fat deposition in piglets and increased the level of CLA supplementation. The serum was detected by biochemical analyzer. The results showed that CLA could treat glucose, triglyceride and triglyceride in serum of piglets. Low density lipoprotein cholesterol (LDL-C) and high density lipoprotein cholesterol (HDL-C) had no obvious effect on the biochemical indexes such as P0.05, but the total cholesterol content was significantly decreased. The immunofluorescence staining of adipose tissue sections showed that the piglets treated with CLA were in the piglets. Fat cells in the back showed obvious signs of apoptosis. Including chromatin aggregation, nuclear condensation DNA fragmentation and other apoptotic characteristics, Especially in 2% CLA group, the apoptotic characteristics of adipocytes were more obvious. The results of enzyme activity test showed that the activities of Caspase-3, Caspase-3 and -9 in dorsal adipose tissue of piglets were significantly increased by adding CLA to diet. Fluorescence quantitative PCR and western blotting were used to detect the apoptosis of adipocytes in piglets. The results showed that CLA treatment down-regulated the expression of mRNA and protein of Bcl2. Increased the expression of mRNA and protein of apoptosis-promoting factor Bax and p53, At the same time, the increase of mRNA expression of Cyt-C and the up-regulation of mRNA expression of Cyt-C protein from mitochondria to cytoplasm. Caspase-3 and the increase of shearing Caspase-3 protein level indicate that Caspase-3 is activated and mediates the apoptosis of adipocytes. On the other hand, the mRNA expression and protein level of TNF- 伪 and its receptor TNFR1 in adipocytes were increased after treatment. These results suggest that both mitochondrial pathway and death receptor pathway are involved in the apoptosis of pig dorsal adipocytes induced by CLA. Dietary supplementation of CLA could reduce back fat accumulation in piglets by inducing adipocyte apoptosis and mitochondrial pathway and death receptor pathway were involved in apoptosis signal transduction.
【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S828.5

【参考文献】